NF-κB or nuclear factor κB is a transcription factor that induces the expression of a large number of pro-inflammatory and anti-apoptotic genes. These include cytokines such as IL-1, IL-2, TNF-α and IL-6, chemokines including IL-8 and RANTES, as well as other pro-inflammatory molecules including COX-2 and cell adhesion molecules such as ICAM-1, VCAM-1, and E-selectin. The NF-κB family includes homo- and heterodimeric transcription factors composed of members of the Rel family (see for example P. A. Baeurle and D. Baltimore, Cell, 1996, 87, 13). Under resting conditions, NF-κB is present in the cytosol of cells as a complex with IκB. The IκB family of proteins serve as inhibitors of NF-κB, interfering with the function of its nuclear localization signal (see for example U. Siebenlist et al., Ann. Rev. Cell Biol., 1994, 10, 405). Upon disruption of the IκB-NF-κB complex following cell activation, NF-κB translocates to the nucleus and activates gene transcription. Disruption of the IκB-NF-κB complex and subsequent activation of NF-κB is initiated by degradation of IκB.
Upon cellular activation by a variety of pro-inflammatory stimuli including IL-1, TNF-α and LPS (bacterial lipopolysaccharide), two specific serine residues of IκB are phosphorylated. Upon phosphorylation, IκB undergoes polyubiquination and subsequent degradation by the 26S proteasome (see for example V. J. Palombella et al., Cell, 1994, 78, 773), freeing NF-κB to translocate to the nucleus. The phosphorylation of IκB is carried out by the IκB kinases (see for example a review by M. Karin and M. Delhase, Seminars in Immunology, 2000, 12, 85). The traditional IKK complex includes at least three subunits, IKKα (also called IKK-1), IKKβ (or IKK-2) and IKKγ (or NEMO), although other relevant complexes involving IKKα and IKKβ may exist. IKKα and IKKβ are both catalytic subunits while IKKλ is believed to be a regulatory subunit. Both IKKα and IKKβ can phosphorylate IκB. For the purposes of this document, the terms IKK or IKK complex refers to any complex that has kinase activity derived from IKKα and/or IKKβ subunits.
In vivo, activation of IKK occurs upon phosphorylation of its catalytic subunit. Both IKKα and IKKβ can be phosphorylated on serine residues, S177 and S181 of the activation loop in the case of IKKβ, and S176 and S180 of the activation loop for IKKα. An IKKβ mutant having alanines in place of serines at 177 and 181 prevented IKKβ phosphorylation and subsequent activation of the IKK complex by TNFα, IL-1 and other upstream activators. These results support a key role for IKKβ in phosphorylation of IκB following proinflammatory stimulation.
Studies in which the NF-κB pathway has been inhibited in cells and animals support the concept that inhibition of the phosphorylation of IκB is a viable approach to treatment of inflammatory, autoimmune and other diseases. In these studies, NF-κB activation was prevented by expression of a non-degradable version of the IκB protein. Expression of this inhibitor in synovial cells derived from rheumatoid arthritis patients reduced the expression of TNF-α, IL-6, IL-1β and IL-8 while the anti-inflammatory molecules IL-10, IL-Ira and IL-11 were not affected. Matrix metalloproteinases (MMP1 and MMP3) were also down-regulated (J. Bonderson et al., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 5668). Transgenic expression of the IκB inhibitor in T cells caused a significant reduction in the severity and onset of collagen-induced arthritis in mice (R. Seetharaman et al., J. Immunol. 1999, 163, 1577). These experiments indicate that suppression of NF-κB in the diseased joint could reduce both the severity and progression of RA. In primary intestinal epithelial cells, the NF-κB inhibitor blocked the expression of IL-1, IL-8, iNOS and COX-2, mediators that are up-regulated during the course of inflammatory bowel disease (C. Jubin et al., J. Immunol., 1998, 160, 410). Expression of this inhibitor in certain tumor cells enhances killing of these cells by chemotherapeutic reagents (A. A. Beg and D. Baltimore, Science, 274, 782). Collectively, the studies described above provide support that inhibition of NF-κB function through inhibition of IKK may be a useful therapeutic approach to treatment of autoimmune and inflammatory disease, and other diseases including cancer.
These results have been confirmed in mice with targeted disruption of the IKKβ gene. Knockout of the IKKβ gene resulted in embryonic lethality due to apoptosis of hepatocytes. However, fibroblasts from the IKKβ knockouts did not undergo IKK and NF-κB activation upon stimulation with IL-1 or TNFα (Q. Li et al., Science, 1999, 284, 321), supporting a key role for IKKβ in and NF-κB activation following inflammatory stimuli.
A conditional knockout was generated by expressing a liver-specific inducible dominant negative IκBβ transgene (I. Lavon et al., Nature Medicine, 2000, 6, 573). These mice were viable with no signs of liver dysfunction even after one year but they did have impaired immune function. This study supports the idea that inhibition of IKKβ can result in immune suppression without damage to the liver.
IKKα knockout mice died shortly after birth and displayed a variety of skeletal defects and skin abnormalities. Fibroblast and thymocytes from these mice showed normal IKK activation and IκB degradation in response to TNFα, IL-1 or LPS (Y. Hu et al., Science, 1999, 284, 316; K. Takeda et al., Science, 1999, 284, 313). Recent studies with knockout and knockin mice have revealed distinct roles for IKKα in development and cell signaling. In contrast to the studies with IKKα knockout mice, mice having a kinase inactive version of IKKα knocked in are viable and fertile, indicating that the perinatal lethality and abnormalities seen in the IKKα knockout mice are not due to the lack of kinase activity. However, these mice do have defects in B cell maturation and development of secondary lymphoid organs (U. Senftleben et al., Science, 2001, 293, 1495). This phenotype appears to be due to a defect in processing of the NF-κB2/p 10 protein to p52, the DNA binding form of this member of the Rel family of transcription factors. In turn, this leads to a defect in the activation of a subset of NF-κB target genes in B cells. In addition, other studies with these same mice have shown that IKKα kinase activity is required for NF-κB activation in the mammary epithelium during pregnancy (Cao, Y., et. al., Cell, 2001, 107,763). This pathway is specifically activated through the TNF receptor family member RANK, requires phosphorylation of the canonical IKK substrate IκBα, and culminates in induction of the cell cycle regulatory gene Cyclin D1.
These studies indicate that an inhibitor of IKKα kinase activity may be useful in treating diseases associated with inappropriate B cell activation such as lupus (O. T. Chan et al., Immunological Rev., 1999, 169, 107) and rheumatoid arthritis (A. Gause and C. Borek, Biodrugs, 2001, 15, 73). In addition, an inhibitor of IKKα may be useful in the treatment of breast cancer since NF-κB is constitutively active in a number of breast tumors and many of these tumors depend on Cyclin D1 for proliferation.
Some inhibitors of IKKβ have been reported. WO 01/58890 describes heteoaromatic carboxamide derivatives as inhibitors of IKKβ. WO 01/68648 describes substituted β-carbolines having IKKβ inhibiting activity. Substituted indoles having IKKβ inhibitory activity are reported in WO 01/30774. WO 01/00610 describes substituted benzimidazoles having NF-κB inhibitory activity. Aspirin and salicylate have been reported to bind to and inhibit IKKβ (M. Yin et al., Nature, 1998, 396, 77).
Substituted pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines having PI3K inhibiting activity are reported in US 2002/0151544 A1. A. J. Bridges described a fused tricylic system, including pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines in the broadest sense, as tyrosine kinase inhibitors (WO9519970). Similarly, J. P. Daub also described a fused ring system, in its broad form encompassing pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines, as fungicides (WO9314080). J. M. Quitela et al (Bioorg. Med. Chem., 1998, 6, 1911) reported that certain substituted pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines could induce or inhibit the release of histamine from rat mast cells. LM-2616, 2,7,9-trimethyl-4-(N-methyl-piperazino)pyrido[3′,2′:4,5]-thieno[3,2-d]pyrimidine, was reported by T. S. Shah et al. as a beta-1 adrenoceptor antagonist and a beta-2 adrenoceptor agonist (Pharm. Comm. 1995,5, 253). Possible antimicrobial activity of compounds with this core structure was reported by J. M. Michael et al. (Al-Azhar, Bull. Science, 1992, 3, 767).
A number of substituted pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines have been described in the chemical literature. Examples include 9-(3-pyridinyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 9-(2-furanyl)-7-phenyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin -4-amine, 9-(4-fluorophenyl)-7-(2-thienyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine -2,4-diamine monohydrobromide, 1-(4-amino-7-methylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin -8-yl)-ethanone, 7-butyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 9-(4-chlorophenyl)-7-(2-thienyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 9-phenyl-7-(2-thienyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 9-(2-chlorophenyl)-7-(2-thienyl) -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7,9-diphenyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7-(4-methoxyphenyl)-9-phenyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 9-methyl-7-phenyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7-(2-thienyl)-9-(trifluoromethyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 7-(4-methoxyphenyl)-9-(trifluoromethyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 9-(4-chlorophenyl)-7-(2-thienyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 9-(4-fluorophenyl)-7-(2-thienyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 7-(2-thienyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 9-phenyl-7-(2-thienyl) -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 7-phenyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 1-(2,4-diamino-7-methylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-8-yl)-ethanone, 2,4,7-triamino-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-8-carbonitrile, 7-methyl-9-(trifluoromethyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7,9-di-2-thienyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 7-ethyl-8-methyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7,8,9-trimethyl-pyrido[3′,2′:4,5]thieno [3,2-d]pyrimidin-4-amine, 7-(2-methylpropyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7-methyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7-cyclopropyl-9-(4-methoxyphenyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 9-(2,4-dichlorophenyl)-7-(2-thienyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7-methyl-9-(trifluoromethyl) -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 4,7-diamino-9-methyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-8-carbonitrile, 2-amino-7-ethoxy-9-phenyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-8-carbonitrile, 2,4,7-triamino-9-(methylthio) -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-8-carbonitrile, 9-(2-furanyl)-7-methyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 7-propyl-pyrido[3′,2′:4,5]thieno [3,2-d]pyrimidin-4-amine, 8-ethyl-7-methyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7,9-bis(trifluoromethyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 7-phenyl-9-(trifluoromethyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 7-phenyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 7,9-dimethyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 7-methyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 9-(4-chlorophenyl)-7-(4-methylphenyl) -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 7,9-diphenyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4-diamine, 4-amino-6,7-dihydro-7-oxo-9-phenyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-8-carbonitrile, 7-(3-pyridinyl) -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7-phenyl-9-(trifluoromethyl) -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7-methyl-9-phenyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7-(4-methylphenyl)-9-phenyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7-(4-fluorophenyl)-9-phenyl -pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine, 7,9-dimethyl-pyrido[3′,2′:4,5]thieno [3,2-d]pyrimidine-4-amine, 7,9-dimethyl-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine.
Some substituted pyrido[3′,2′:4,5]thieno[3,2-d][1,2,3]triazines were described by J. M. Quintela et al (Eur. J. Med. Chem., 1998, 33, 887) as antihistamines. A number of these compounds were cytotoxic against several human and mouse tumor cell lines. Some other substituted pyrido[3′,2′:4,5]thieno[3,2-d][1,2,3]triazines with antimicrobial activity were reported by F. Guerrera et al (Farmaco, 1993, 48, 1725). D. Y. Raymond described some substituted pyrido[3′,2′:4,5]thieno[3,2-d][1,2,3]triazines as anti-allergy agents (U.S. Pat. No. 4,239,887). A number of substituted pyrido[3′,2′:4,5]thieno[3,2-d][1,2,3]triazines were reported in the literature, including: 4-(4-methyl-1-piperazinyl)-7,9-diphenyl-pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazine, 1-[4-[4-(7,9-diphenylpyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-yl)-1-piperazinyl]phenyl]-ethanone, N-(4-morpholinylmethyl)-pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-amine, N-methyl-pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-amine, N -butyl-7-methyl-4-(1-piperidinyl)-pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazine-9-carboxamide, N-butyl-4-[[(2-chlorophenyl)methyl]amino]-7-methyl -pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazine-9-carboxamide, N-butyl-4-[[2-(diethylamino)ethyl]amino]-7-methyl-pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazine-9-carboxamide, N-butyl-4-(butylamino)-7-methyl-pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazine-9-carboxamide, 2-[[7-methyl-9-(4-pyridinyl)pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-yl]amino]-ethanol, 2-[[7-methyl-9-(3-pyridinyl)pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-yl]amino]-ethanol, N,N-dimethyl-N′-[7-methyl-9-(4-pyridinyl)pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-yl]-1,2-ethanediamine, N,N -dimethyl-N′-[7-methyl-9-(3-pyridinyl)pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-yl]-1,2-ethanediamine, 9-(4-chlorophenyl)-7-phenyl-4-(1-piperidinyl)-pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazine, 7-methyl-N-[3-(4-morpholinyl)propyl]-pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-amine, 7-methyl-pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazine, 7-methyl -pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4(1H)-one (1-methylethylidene)hydrazone, 2-(7-methylpyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-yl)-hydrazinecarboxylic acid methyl ester, 7-methyl-pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-amine, and N,7-dimethyl -pyrido[3′,2′:4,5]thieno[3,2-d]-1,2,3-triazin-4-amine.
Substituted pyrido[3′,2′:4,5]thieno[3,2-d][1,2,3]pyridines have been reported in the literature (Baba et al, Chem. Pharm. Bull., 1999, 47, 993).